Antibacterial antibiotics LL-CO8078α1, α2, α3 and β

ABSTRACT

This disclosure describes four new antibacterial agents designated LL-CO8078α 1 , LL-CO8078α 2 , LL-CO8078α 3  and LL-CO8078β produced in a microbiological fermentation under controlled conditions using a new strain of a new species of the genus Streptomyces called Streptomyces majorciensis Labeda, sp. nov., and mutants thereof. These new antibacterial agents are active against a variety of microorganisms and thus are useful in inhibiting the growth of such bacteria wherever they may be found. In addition, these agents are active as growth promotants in warm-blooded animals.

BRIEF SUMMARY OF THE INVENTION

This invention relates to four new antibacterial agents designatedLL-CO8078α₁, LL-CO8078α₂, LL-CO8078α₃ and LL-CO8078β; to theirproduction by fermentation, to methods for their receovery andconcentration from crude solutions and to processes for theirpurification. The present invention includes within its scope theantibacterial agents in dilute forms, as crude concentrates and in purecrystalline forms. The effects of the new antibacterial agents onspecific microorganisms, together with their chemical and physicalproperties, differentiate them from previously described antibacterialagents.

The molecular structure of these antibacterial agents is unknown at thepresent time.

DETAILED DESCRIPTION OF THE INVENTION

The new antibacterial agents designated LL-CO8078α₁, LL-CO8078α₂,LL-CO8078α₃ and LL-CO8078β are formed during the cultivation undercontrolled conditions of a new strain of a new species Streptomycesnamed Streptomyces majorciensis Labeda, sp. nov. This new antibioticproducing strain is maintained in the culture collection of the LederleLaboratories Division, American Cyanamid Company, Peral River, N.Y. asculture number LL-CO8078. A viable culture of the new microogranism hasbeen deposited with the Patent Culture Collection, FermentationLaboratory, Northern Regional Research Center, United States Departmentof Agriculture, Peoria, Ill., and has been added to its permanentcollection. It is freely available to the public from this depositoryunder its accession number NRRL 15167.

The new antibiotics LL-CO8078α₁ and LL-CO8078β have now been discoveredto be members of the Aurodox family of antibiotics [Can. J. Chem., 58:501-526 (1980)] but are the only members other than antibiotic A40A(U.S. Pat. No. 4,264,407) that contain a tetraene as a part of thechromophore. LL-CO8078α₁ and A40A have optical rotations of oppositesign and differ significantly in their ¹³ C NMR spectra in the 95-115ppm region when the spectra are run in methanol. LL-CO8078β shows asignificant difference from A40A in optical rotation and has a ¹³ C NMRspectrum very similar to LL-CO8078α₁.

The following is a comparison of Aurodox, LL-CO8078α₁ and LL-CO8078β ¹³C NMR chemical shifts. The ¹³ C NMR spectra (20 MHz) of the antibioticsin the free acid form were run in DMSO-d₆ with tetramethylsilane (TMS)as an internal reference. Peak positions are given below in parts permillion from TMS. Not all overlapping peaks are indicated forLL-CO8078α₁ and LL-CO8078β.

    ______________________________________                                        Aurodox     LL-CO8078α.sub.1                                                                    LL-CO8078β                                       ______________________________________                                        195.6       195.2       195.2                                                 175.4       174.0       171.6                                                 163.0       163.2       163.2                                                 160.0       160.8       160.8                                                 139.9       140.4       140.5                                                 139.9       140.1       140.5                                                 139.7       140.1       140.1                                                 136.3       137.6       137.6                                                 135.6       135.7       135.7                                                 134.6       133.1       135.3                                                 130.9       132.7       --                                                    129.9       129.8       130.8                                                 129.9       129.8       130.1                                                 129.0       129.0       129.1                                                 128.3       128.1       128.2                                                 127.5       127.8       --                                                    126.0       126.0       126.0                                                 125.2       125.9       126.0                                                 124.6       125.1       124.9                                                 109.4       110.7       110.7                                                 99.4        97.9        97.4                                                  98.3        98.7        98.6                                                  89.0        87.7        87.8                                                  80.1        --          --                                                    79.2        --          --                                                    74.9        75.4        75.7                                                  72.7        --          --                                                    72.1        --          --                                                    71.3        71.0        71.0                                                  69.3        69.9        70.3                                                  --          67.3        67.4                                                  55.2        55.2        58.5                                                  49.2        55.4        55.5                                                  40.0        40.0        40.0                                                  38.1        38.6        38.8                                                  35.6        35.9        35.8                                                  34.5        35.2        35.2                                                  23.8        22.4        22.4                                                  19.3        20.2        19.5                                                  15.3        --          --                                                    12.8        13.0        13.1                                                  12.1        11.9        12.2                                                  11.4        11.8        11.8                                                  10.9        11.1        11.1                                                  10.6        10.5        10.5                                                  --           8.8         8.8                                                  ______________________________________                                    

Culture LL-CO8078, which produced a novel antibiotic with rumen growthpromotant activity, was isolated from a soil sample collected inMajorca, Spain. The culture was taxonomically characterized and wasidentified as a new species of the red-series Streptomyces to be knownas Streptomyces majorciensis Labeda, sp. nov. Observations were made ofthe cultural, physiological and morphological features of the culture inaccordance with methods detailed by E. B. Shirling and D. Gottlieb,Methods for characterization of Streptomyces species. Internat. J. Syst.Bacteriol. 16: 313-340 (1967). Media used in this study were selectedfrom those recommended by T. G. Pridham, et al., A selection of mediafor maintenance and taxonomic study of Streptomycetes. Antibiotics Ann.pp. 947-953 (1956-57), for the taxonomic study of actinomycetes.Chemical composition of the cell walls of the culture was determined byusing the method of H. A. Lechevalier, et al., Chemical composition as acriterion in the classification of Actinomycetes. Adv. Appl. Microbiol.14: 47-72 (1971). Details are recorded in Tables I-IV, and a generaldescription of the culture is given below. Underscored descriptivecolors are taken from K. L. Kelly and D. B. Judd, Color. UniversalLanguage and Dictionary of Names. Nat. Bur. Stand. (U.S.) Spec. Publ.440, Washington, D.C. (1976) and the accompanying Inter-Society ColorCouncil, Nat. Bur. Stand. Centroid Color Charts.

MICROMORPHOLOGY

Spores are formed in long straight chains (Rectus flexibilis) on aerialsporophores. The spores are phlangiform (0.8-0.9 micron by 1.3-1.4micron) and the surface of the mature spores is smooth when observed byscanning electron microsocpy.

CELL WALL COMPOSITION

Whole cell hydrolysates of this culture contain the L,L-isomer ofdiaminopimelic acid, placing it in the Type I cell wall group ofLechevalier, et al., (vide supra). This is typical of all Streptomycesspecies.

AMOUNT OF GROWTH

Good growth is observed on all media.

AERIAL MYCELIUM AND SPORE COLOR

Aerial mycelium is white; spore masses are pinkish gray shades, rangingfrom 10. pinkish gray to 8. grayish pink. Sporulation is heavy to veryheavy, depending on the medium.

SOLUBLE PIGMENTS

Absent on many media; brownish shades where produced.

REVERSE COLOR

Yellow to yellowish brown shades on all media.

PHYSIOLOGICAL REACTIONS

Nitrates not reduced to nitrites; weak liquification of gelatin in 14days; black pigment produced on peptone-yeast extract-iron agar but noton tyrosine medium in 7 days. Carbohydrate utilization as per the methodof T. G. Pridham and D. Gottlieb, the utilization of carbon compounds bysome Actinomycetales as an aid for species determination, J. Bacteriol.56:107-114 (1948): good utilization of galactose, glucose, glycerol,maltose, mannose and trehalose; poor utilization of fructose andsalicin; no utilization of adonitol, arabinose, dulcitol, inositol,lactose, mannitol, melezitose, melibiose, raffinose, rhamnose, sorbitol,sucrose, xylose.

Culture LL-CO8078 was compared with Streptomyces reference cultures fromthe aforesaid Lederle culture collection which are known to produceantibiotics of this class and a reference culture of the red-sporedstreptomycete group closest to this strain. The following observationswere made of 14-day growth on yeast extract-malt extract agar:

    ______________________________________                                                     Spore Mass  Soluble  Reverse                                     Culture      Color       Pigments Color                                       ______________________________________                                        RC47 S. goldiensis                                                                         light gray  none     pale yellow                                 ATCC 21386                                                                    RC52 S. filipinensis                                                                       light gray  none     pale yellow                                 NRRL11044                                                                     RC97 S. xanthophaeus                                                                       no sporulation                                                                            none     yellowish                                   NRRLB5414                         white                                       LL-C08078 S. major-                                                                        pinkish gray                                                                              none     yellow brown                                ciensis                                                                       ______________________________________                                    

Culture LL-CO8078 resembles none of the Streptomyces species producingantibiotics of the mocimycin class. Moreover, it resembles no describedspecies of the genus Streptomyces, and thus, based on the observationspresented, a new species is proposed to be called Streptomycesmajorciensis Labeda, sp. nov.

                                      TABLE I                                     __________________________________________________________________________     Cultural Characteristics of LL-C08078                                        Incubation: 14 Days   Temperature: 28° C.                              Medium Amount of Growth                                                                        Aerial Mycelium and/or Spores                                                                    Soluble Pigment                                                                        Reverse color                    __________________________________________________________________________    Glycerol-                                                                            Good      Plicate growth with heavy                                                                        None     Pale yellow                      Asparagine                                                                           to        sporulation; spore mass 10. pinkish                          Agar   Moderate  gray.                                                        Hickey-                                                                              Good      Raised, mounded colonies; Sporulation                                                            Brownish Dark yellowish                   Tresner          heavy; spore masses 10. pinkish gray                                                                      brown                            Agar             with tufts of white aerial mycelia.                          Inorganic                                                                            Good      Heavy sporulation; spore                                                                         Brownish Pale yellow                      Salts-Starch     mass 10. pinkish gray.                                       Agar                                                                          Oatmeal                                                                              Good      Heavy sporulation; 10. pinkish                                                                   None     Pale yellow                      Agar             gray to 8. grayish pink;                                                      white tufts of aerial mycelia.                               Tomato Paste                                                                         Good      Very heavy sporulation on raised                                                                 Brownish --                               Oatmeal          colonies; spore mass 10. pinkish                             Agar             gray; scattered tufts of white aerial                                         mycelia.                                                     Yeast Extract                                                                        Good      Heavy sporulation; 10.                                                                           None     Strong yellowish                 Malt Extract     pinkish gray.               brown                            Agar                                                                          __________________________________________________________________________

                                      TABLE II                                    __________________________________________________________________________    Micromorphology of LL-C08078                                                  Medium Aerial Mycelium and/or Sporiferous Structures                                                        Spore Shape                                                                          Spore Size                                                                            Spore Surface                    __________________________________________________________________________    Yeast Extract                                                                        Spore chains arise as straight chains from aerial                                                    phlangiform                                                                          0.8-0.9 micron                                                                        Smooth                           Malt Extract                                                                         sporophores (Rectus flexibilis)                                                                             ×                                  Agar                                 1.3-1.4 micron                           __________________________________________________________________________

                  TABLE III                                                       ______________________________________                                        Physiological Reactions of LL-C08078                                                  Incubation  Amount of Physiological                                   Medium  Period (Days)                                                                             Growth    Reaction                                        ______________________________________                                        Peptone -                                                                             7           Good      Blackening                                      Iron Agar                                                                             14          Good      Blackening                                      Tyrosine                                                                              7           Good      No pigment                                      Agar    14          Good      No pigment                                      Litmus  7           Good      Moderate proteolysis                            Milk    14          Good      Strong proteolysis                              Nutrient                                                                              7           Good      No proteolysis                                  Gelatin 14          Good      Weak proteolysis                                Nitrate 14          Good      No reduction                                    Broth                                                                         Esculin 7           Good      Hydrolysis                                      Broth                                                                         Urea    14          Good      No hydrolysis                                   Broth                                                                         ______________________________________                                    

                  TABLE IV                                                        ______________________________________                                         Carbon Source Utilization of LL-CO8078                                       Incubation: 14 Days  Temperature: 28° C.                               Carbon Source:  Utilization*                                                  ______________________________________                                        Adonitol        0                                                             1-Arabinose     0                                                             Dulcitol        0                                                             Fructose        1                                                             d-Galactose     3                                                             d-Glucose       3                                                             Glycerol        3                                                             i-Inositol      0                                                             Lactose         0                                                             Maltose         3                                                             Mannose         3                                                             d-Mannitol      0                                                             Melezitose      0                                                             Melibiose       0                                                             d-Raffinose     0                                                             1-Rhamnose      0                                                             Salicin         1                                                             Sorbitol        0                                                             Sucrose         0                                                             Trehalose       3                                                             Xylose          0                                                             Negative control                                                                              0                                                             ______________________________________                                         *3 = Good utilization                                                         2 = Fair utilization                                                          1 = Poor utilization                                                          0 = No utilization                                                       

It is to be understood that for the production of these newantibacterial agents, the present invention is not limited to thisparticular organism or to organisms fully answering the above growth andmicroscopic characteristics, which are given for illustrative purposesonly. In fact, it is desired and intended to include in the term"Streptomyces majorciensis Labeda, sp. nov., NRRL 15167" the natural(spontaneous) mutants of this organism as well as induced mutantsproduced from this organism by various mutagenic means known to thoseskilled in the art, such as exposure to X-ray radiaton, ultravioletirradiation, nitrogen mustard, actinophages, nitrosamines and the like.It is also desired and intended to include inter- and intra-specificgenetic recombinants produced by genertic techniques known to thoseskilled in the art, such as, for example, conjugation transduction, andgenetic engineering techniques.

The antibacterial agents were tested in vitro using a variety ofgram-positive bacteria by the standard agar dilution procedure. Theresults are reported as minimal inhibitory concentrations (mcg/ml) inTable V.

                  TABLE V                                                         ______________________________________                                        In Vitro Antibacterial Activity                                                         Minimal Inhibitory Conc. (mcg/ml)                                   Organism    LL-C08078α.sub.1                                                                   LL-C08078α.sub.3                                                                   LL-C08078β                             ______________________________________                                        Streptococcus                                                                             8          8          32                                          β-hemolytic C203                                                         Streptococcus                                                                             16         256        64                                          β-hemolytic                                                              636TCR                                                                        Streptococcus                                                                             16         256        128                                         β-hemolytic AGB                                                          Sarcina lutea                                                                             128                   128                                         PCI1001                                                                       Corynebacterium                                                                           256        128        128                                         minutissimum                                                                  LL No. 151                                                                    ______________________________________                                    

These compounds are also active in vivo as evidenced by theireffectiveness against lethal infections in warm-blooded animals.LL-CO8078α₁ is active when administered subcutaneously to mice whichhave been infected with a lethal dose of Streptococcus pyogenes C203.The ED₅₀ for LL-CO8078α₁ is 131 mg/kg of body weight.

These compounds are also active in vivo as growth promoting agents inwarm-blooded animals.

FERMENTATION PROCESS

Cultivation of Streptomyces majorciensis Labeda, sp. nov. NRRL 15167 maybe carried out in a wide variety of liquid culture media. Media whichare useful for the production of these novel antibacterial agentsinclude an assimilable source of carbon such as starch, sugar, molasses,glycerol, etc.; an assimilable source of nitrogen such as protein,protein hydrolysate, polypeptides, amino acids, corn steep liquor, etc.;and inorganic anion and cation salts, such as potassium, sodium,ammonium, calcium, sulfate, carbonate, phosphate, chloride, etc. Traceelements such as boron, molybdenum, copper, etc., are supplied asimpurities of other constituents of the media. Aeration in tanks,bottles and flasks is supplied by forcing sterile air through or ontothe surface of the fermenting medium. Further agitation in tanks isprovided by a mechanical impeller. An antifoaming agent such as lard oilor silicone defoamer may be added as needed.

INOCULUM PREPARATION

Shaker flask inoculum of Streptomyces majorciensis Labeda, sp. nov. NRRL15167 is prepared by inoculating 100 ml or 200 ml portions of sterileliquid medium in appropriate flasks with scrapings or washings of sporesfrom an agar slant of the culture. The following is an example of asuitable medium:

    ______________________________________                                        Corn starch       1.2%                                                        Dextrose          0.6%                                                        Beef extract      0.3%                                                        Yeast extract     0.5%                                                        tryptone.sup.1    0.5%                                                        Calcium carbonate 0.2%                                                        Water qs          100%                                                        ______________________________________                                         The pH is adjusted to 7.5 with an alkali metal hydroxide and the mixture      is sterilized prior to inoculation.                                           [.sup.1 A peptone, registered trademark of Difco Laboratories, Detroit,       Michigan]-                                                               

These flasks are incubated at 25°-29° C., preferably 28° C. and agitatedat 180 r.p.m. on a rotary shaker for 30-50 hours. This inoculum is thenused to inoculate one liter or 12 liter batches of the same sterileinoculum at the rate of 100 ml per liter or 200 ml per 12 liters, inglass bottles. This bottle inoculum is aerated by a sterile air flow of10-40 liters per minute while growth is continued at 25°-29° C.,preferably 28° C., for 24-50 hours.

These bottle inocula are then used to inoculate 260 liters of the samesterile media in 300 liter tanks. The tank inoculum is grown at 25°-29°C., preferably 28° C., with sterile air flow of 100-200 liters perminute and agitation at 200-300 r.p.m. for 24-50 hours and then used toinoculate tank fermentors.

TANK FERMENTATION

For the production of these antibacterial agents in tank fermentors thefollowing sterilized medium may be used:

    ______________________________________                                        Dextrin             5.0%                                                      Dextrose            0.5%                                                      Soy flour           3.5%                                                      Calcium carbonate   0.7%                                                      Cobalt chloride hexahydrate                                                                       0.00025%                                                  Water qs            100%                                                      ______________________________________                                    

Each tank is inoculated with 3-10% of the tank inoculum described above.Aeration is supplied at the rate of 0.2-0.8 liter of sterile air perliter of media per minute and the fermenting mash is agitated by animpeller driven at 100-200 r.p.m. The temperature is maintained at25°-29° C., usually 28° C. and the fermentation is normally continuedfor 75-100 hours, at which time the mash is harvested.

This invention will be described in greater detail in conjunction withthe following non-limiting examples.

EXAMPLE 1

    ______________________________________                                        Inoculum Preparation                                                          A typical medium used to grow the primary                                     inoculum was prepared according to the following formula:                     ______________________________________                                        Corn starch       1.2%                                                        Dextrose          0.6%                                                        Beef extract      0.3%                                                        Yeast Extract     0.5%                                                        tryptoneTM.       0.5%                                                        Calcium carbonate 0.2%                                                        Water qs          100%                                                        ______________________________________                                    

The pH was adjusted to 7.5 with 6N sodium hydroxide and the medium wassterilized at 121° C. for 15 minutes. A 100 ml portion of this sterilemedium, in a flask, was inoculated with scrapings from an agar slant ofthe culture Streptomyces majorciensis Labeda, sp. nov. NRRL 15167. Themedium was placed on a rotary shaker and agitated vigorously at 180r.p.m. for 48 hours at 28° C.

The resulting primary inoculum was used to inoculate one liter of thesame sterile medium in a 2-liter bottle, which was aerated with sterileair flow of 10 liters per minute and grown at 28° C. for 48 hours,providing secondary inoculum.

Two one-liter portions of this secondary inoculum were used to inoculate260 liters of the same sterile medium in a 300-liter tank. This tankinoculum was grown at 28° C. with sterile air flow of 150 liters perminute and agitation by an impeller driven at 230 r.p.m. for 24 hours,providing tertiary (tank) inoculum.

EXAMPLE 2

    ______________________________________                                        Fermentation                                                                  A fermentation medium was prepared according to                               the following formula:                                                        ______________________________________                                        Dextrin             5.0%                                                      Dextrose            0.5%                                                      Soy flour           3.5%                                                      Calcium carbonate   0.7%                                                      Cobalt chloride hexahydrate                                                                       0.00025%                                                  Water qs            100%                                                      ______________________________________                                    

A 2800 liter portion of this medium was sterilized at 121° C. for 60minutes and then inoculated with 260 liters of the tertiary inoculumdescribed in Example 1. After sterilization, pH is 7.0. Aeration wassupplied at the rate of 1650 liters of sterile air per minute andagitation was supplied by an impeller driven at 100 r.p.m. Thetemperature was maintained at 28° C. and the fermentation was terminatedafter 88 hours at which time the mash was harvested.

EXAMPLE 3 Preliminary Isolation of LL-CO8078α₁, α₂, α₃ and β

A harvest mash, prepared as described in Example 2, comprising 2950liters was extracted with 1125 liters of methylene chloride. The organicextract was then concentrated in vacuo to a residue, giving 767 g (SolidA). The remaining aqueous portion was reextracted with 1000 liters offresh methylene chloride and this organic extract was concentrated invacuo to a residue, giving 646 g (Solid B).

The solid (A) was suspended in a mixture of 2 parts ether and one parthexane and shaken gently. The mixture was allowed to separate and theliquid portion was removed by decantation. The residue was dissolved inmethylene chloride and then concentrated in vacuo to a residue, giving241 g (Solid C).

A harvest mash, prepared as described in Example 2, comprising 3000liters, was extracted with 1200 liters of methylene chloride. Theorganic extract was concentrated in vacuo to a residue, giving 426 g(Solid D).

The solids B, C and D were combined (total weight 1313 g) and suspendedin ether. This suspension was filtered and the solid was washed withether and dried, giving 309 g of combined components LL-CO8078α₁, α₂, α₃and β.

EXAMPLE 4 Isolation of LL-CO8078α₁

A glass column with a diameter of 3 inches was filled to a height of 20inches with silica gel. A 12 g portion of the product of Example 3 wasstirred with 100 ml of ethyl acetate, filtered and the filtrate allowedto seep into the column. The column was devedloped with 1.2 liters ofethyl acetate and then with ethyl acetate containing 5% of absoluteethanol. Fractions of 75 ml each were collected and monitored foractivity by bioautography against B. cereus. Fractions 72-120 werecombined and concentrated in vacuo, giving 2.311 g of a solid.

A glass column with a diameter of 9 inches was packed to a height of 20inches with silica gel. A 218 g portion of the product of Example 3 wasstirred with 2 liters of ethyl acetate, filtered and the filtrateallowed to seep into the column. The charge was washed in with 13 litersof fresh ethyl acetate and the column was then developed with ethylacetate containing 8% ethanol. Fractions of 4 liters each were collectedand checked for activity as described above, then the column wasstripped with 50 liters of ethyl acetate:ethanol (8:12). Fractions 1 and2 were saved for use in Example 5. Fractions 11-19 were combined andconcentrted in vacuo to a yellow residue, weighing 24 g.

The above two solids were combined giving 26 g of LL-CO8078α₁, havingthe following characteristics:

Elemental analysis: C, 65.95; H, 8.06; N, 3.21; O, 23.16;

    ______________________________________                                        [α].sub.D.sup.26 =                                                                 -44° ± 2 (0.496% in chloroform)                          =          -89° ± 1 (0.790% in methanol);                           ______________________________________                                    

    ______________________________________                                        UV spectra as shown in FIG. I:                                                                   10 mcg/ml in methanol                                                         10 mcg/ml in 0.1N HCl                                                         10 mcg/ml in 0.1N NaOH;                                    ______________________________________                                    

An IR spectrum in kBr as shown in FIG. II; A ¹³ C NMR Spectrum 20 MHz ind₆ DMSO with reference equivalent to internal TMS standard as shown inFIG. III; A proton NMR Spectrum 80 MHz in d₆ DMSO with internal TMSreference standard as shown in FIG. IV; Molecular weight by massspectroscopy 778.

EXAMPLE 5 Isolation of LL-CO8078α₂ and α₃

A glass column with a diameter of 9 inches was packed to a height of 20inches with silica gel. The fractions 1 and 2 (4 liters each) saved inExample 4 were combined and concentrated in vacuo, giving 42 g of anoily residue. This residue was dissolved in ethyl acetate and charged ona silica gel column. The column was eluted with ethyl acetate, checkingthe fractions for activity by bioautography. Fractions 1-60 werecombined and lyophilized giving 25.0 g of solid. This solid wasdissolved in 20 ml of chloroform and allowed to seep into a 3/4 inch by16 inch column of silica gel. The charge was washed in with 100 ml ofchloroform and then the column was developed first withchloroform:acetone (4:1) (fractions 1-50) and then withchloroform:acetone (3:2). Fractions of 15 ml each were collected andchecked for activity by bioautography.

Fractions 71-75 were combined and concentrated in vacuo to a residuewhich was lyophilized from t-butanol, giving 64 mg of LL-CO8078α₃.

Fractions 79-84 were combined and concentrated in vacuo, giving 99 mg ofLL-CO8078α₂.

LL-CO8078α₃ has the following characteristics: Elemental analysis: C,67.99; H, 8.60; N, 1.14; O, 22.27 (by difference); [α]_(D) ²⁶ =-31°±5(0.163% in methanol);

    ______________________________________                                        UV Spectra as shown in FIG. V:                                                                   10 mcg/ml in methanol                                                         10 mcg/ml in 0.1N HCl                                                         10 mcg/ml in 0.1N NaOH;                                    ______________________________________                                    

An IR spectrm in KBr as shown in FIG. VI; A Proton NMR Spectrum 80 MHzin CDCl₃ with reference equivalent to internal TMS standard as shown inFIG. VII. LL-CO8078α₂, has the following characteristics: Elementalanalysis: C, 65.02; H, 8.03; N, 1.54; O, 25.41 (by difference); [α]_(D)²⁶ =-32°±5 (0.280% in methanol);

    ______________________________________                                        UV Spectra as shown in FIG. VIII:                                                                 10 mcg/ml in methanol                                                         10 mcg/ml in 0.1N HCl                                                         10 mcg/ml in 0.1N NaOH;                                   ______________________________________                                    

An IR Spectrum in KBr as shown in FIG. IX; A Proton NMR Specrum 80 MHzin d₆ DMSO with reference equivalent to internal TMS standard as shownin FIG. X.

EXAMPLE 6 Isolation of LL-CO8078β

A glass column with a diameter of 3 inches was filled to a height of 20inches with silica gel. A 12 g portion of the product of Example 3 wasstirred with 100 ml of ethyl acetate and filtered. The filtrate wasallowed to seep into the column which was then developed first with 1.2liters of ethyl acetate, then with ethyl acetate containing 5% absoluteethanol and collecting a total of 150 fractions of 75 ml each. Thecolumn was then stripped with ethyl acetate:methanol (1:1) and thisstrip was concentrated in vacuo, giving 1.733 g of solid. A 500 mgportion of this solid was dissolved in one ml of chloroform and allowedto seep into a 3/4 inch column packed to a height of 40 cm with silicagel. The charge was washed in with 25 ml of chloroform and the columnwas then developed with chloroform-acetone (1:1) collecting fractions of20 ml each and monitoring for activity by bioautography. Fractions 13-22were combined, desolventized and lyophilized, giving 320 mg ofLL-CO8078β, having the following characteristics: Elemental analysis: C,65.82; H, 7.92; N, 3.62; O, 22.64 (by difference);

    ______________________________________                                        [α].sub.D.sup.26 =                                                                 -29° ± 4 (0.245% in chloroform)                          =          -120° ± 5 (0.175% in methanol);                          ______________________________________                                    

    ______________________________________                                        UV Spectra as shown in FIG. XI:                                                                   10 mcg/ml in methanol                                                         10 mcg/ml in 0.1N HCl                                                         10 mcg/ml in 0.1N NaOH;                                   ______________________________________                                    

An IR Spectrum in KBr as shown in FIG. XII; A Proton NMR Spectrum 80 MHzin d₆ DMSO with reference equivalent to internal TMS standard as shownin FIG. XIII;

A ¹³ C NMR Spectrum 20 MHz in d₆ DMSO with reference equivalent tointernal TMS standard as shown in FIG. XIV.

We claim:
 1. Antibacterial antibiotic LL-CO8078α₁, a composition whichis effective in inhibiting the growth of bacteria and in itssubstantially pure form has:(a) the following elemental analysis(percent): C, 65.95; H, 8.06; N, 3.21; O, 23.16; (b)

    ______________________________________                                        optical rotations [α].sub.D.sup.26 =                                                   -44° ± 2 (0.496% in chloroform);                     =              -89° ± 1 (0.790% in methanol);                       ______________________________________                                    

(c) characteristic ultraviolet absorption spectra as shown in FIG. I ofthe attached drawings; (d) a characterstic infrared absorption spectrumas shown in FIG. II of the attached drawings; (e) a characteristic ¹³C-nuclear magnetic resonance spectrum as shown in FIG. III of theattached drawings; (f) a characteristic proton nuclear magneticresonance spectrum as shown in FIG. IV of the attached drawings; and (g)a molecular weight by mass spectroscopy of
 778. 2. Antibacterialantibiotic LL-CO8078α₂, a composition which is effective in inhibitingthe growth of bacteria and in its substantially pure form has:(a) thefollowing elemental analysis (percent); C, 65.02; H, 8.03; N, 1.54; O,25.41 (by difference); (b) an optical rotation [α]_(D) ²⁶ =-32°±5(0.280% in methanol); (c) characteristic ultraviolet absorption spectraas shown in FIG. VIII of the attached drawings; (d) a characteristicinfrared absorption spectrum as shown in FIG. IX of the attacheddrawings; and (e) a characteristic proton nuclear magnetic resonancespectrum as shown in FIG. X of the attached drawings.
 3. Antibacterialantibiotic LL-CO8078α₃, a composition which is effective in inhibitingthe growth of bacteria and in its substantially pure form has:(a) thefollowing elemental analysis (percent); C, 67.99; H, 8.60; N, 1.14; O,22.27 (by difference); (b) an optical rotation [α]_(D) ²⁶ =-31°±5(0.163% in methanol); (c) characteristic ultraviolet absorption spectraas shown in FIG. V of the attached drawings; (d) a characteristicinfrared absorption spectrum as shown in FIG. VI of the attacheddrawings; and (e) a characteristic proton nuclear magnetic resonancespectrum as shown in FIG. VII of the attached drawings.
 4. Antibacterialantibiotic LL-CO8078β, a composition which is effective in inhibitingthe growth of bacteria and in its substantially pure form has:(a) thefollowing elemental analysis (percent); C, 65.82; H, 7.92; N, 3.62; O,22.64 (by difference); (b) optical rotations

    ______________________________________                                        (b) optical rotations [α].sub.D.sup.26 =                                                -29° ± 4 (0.245% in chloroform);                    =               -120° ± 5 (0.175% in methanol);                     ______________________________________                                    

(c) characteristic ultraviolet absorption spectra as shown in FIG. XI ofthe attached drawings; (d) a characteristic infrared absorption spectrumas shown in FIG. XII of the attached drawings; (e) a characteristicproton nuclear magnetic resonance spectrum as shown in FIG. XIII of theattached drawings; and (f) a characteristic ¹³ C-nuclear magneticresonance spectrum as shown in FIG. XIV of the attached drawings.
 5. Amethod of treating bacterial infections in a warm-blooded animal whichcomprises administering to said animal an effective antibacterial amountof antibiotic LL-CO8079α₁ as defined in claim 1, antibiotic LL-CO8078α₂as defined in claim 2, antibiotic LL-CO8078α₃ as defined in claim 3 orantibioti LL-CO8078β as defined in claim
 4. 6. A therapeutic compositionfor the treatment of bacterial infections in a warm-blooded animal whichcomprises an effective antibacterial amount of an antibacterial agentantibiotic LL-CO8078α₁ as defined in claim 1, antibiotic LL-CO8078α₂ asdefined in claim 2, antibiotic LL-CO8078α₃ as defined in claim 3 orantibiotic LL-CO8078β as defined in claim 4, and a pharmaceuticalcarrier.
 7. A process for preparing antibiotic LL-CO8078α₁ as defined inclaim 1, antibiotic LL-CO8078α₂ as defined in claim 2, antibioticLL-CO8078α₃ as defined in claim 3 or antibiotic LL-CO8078β as defined inclaim 4 which comprises cultivating Streptomyces majorciensis Labeda,sp. nov., having the identifying characteristics of NRRL 15167, ormutants thereof, under aerobic conditions, in a sterile liquid mediumcontaining assimilable sources of carbon, nitrogen and inorganic anionand cation salts, until substantial antibiotic activity is imparted tosaid medium by the production of LL-CO8078α₁, LL-CO8078α₂, LL-CO8078α₃or LL-CO8078β, and then recovering the antibiotic therefrom.
 8. Aprocess for preparing antibiotic LL-CO8078α₁ as defined in claim 1,antibiotic LL-CO8078α₂ as defined in claim 2, antibiotic LL-CO8078α₃ asdefined in claim 3 or antibiotic LL-CO8078β as defined in claim 4 whichcomprises aerobically fermenting a liquid medium containing assimilablesources of carbon, nitrogen and inorganic anion and cation salts, whichmedium has been inoculated with a viable culture of Streptomycesmajorciensis Labeda, sp. nov., having the identifying characteristics ofNRRL 15167, or mutants thereof, maintaining said fermentation culturewith sterile aeration and agitation for a period of 75-100 hours at25°-29° C., harvesting the mash, extracting the crude product inmethylene chloride and purifying by conventional chromatography.